The dissolution of the collagen triple-helix has been implicated in a variety of diseases that effect the structural integrity of various components of the body. Collagen also provides a barrier between tissues and cells; destruction of this barrier plays a role in tumor cell invasion and metastasis. The matrix metalloproteinase (MMP) family has been recognized for their collagenolytic activities, and has thus been the subject of intense research efforts, in order to elucidate their mechanisms of action and allow for rational design of inhibitors. We have developed methodology for constructing triple-helical peptides (THPs) and have applied these synthetic proteins for the study of enzyme interactions with collagens. "Triple-helical peptidase" activity was found to be more widespread amongst proteases than previously believed. It appears that the unique aspect of collagenolytic activity may not be the ability to cleave a triple-helix, but rather the ability to bind and orient the native collagen molecule properly. This paradigm shift could have significant effects on the design of inhibitors against collagenolytic activity. To further explore the nature of triple-helical peptidase activity, a series of THP substrates will be assembled, incorporating known sites of collagen hydrolysis and varying over a range of Tm values. Substrate thermal stability will be correlated to enzyme activity. In tandem, 2D NMR experiments using 15N-labeled amino acids will examine the mobilities of the peptide backbone in these substrates. Individual kinetic parameters and activation energies will be evaluated for MMP, trypsin, thermolysin, cathepsin K, and aggrecanase hydrolysis of THPs. MMP-1, MMP-2, MMP-8, and cathepsin K mutants will be utilized to determine the regions within these enzymes critical for triple-helical peptidase activity. Finally, selective THP substrates and novel THP inhibitors will be designed and tested. Overall, triple-helical peptidase activity will be systematically evaluated for a variety of proteases as a function of substrate sequence and conformational flexibility, and the mechanism of collagenolytic activity will better understood.